The Src family of protein-tyrosine kinases plays a central role in cellular growth, differentiation and function. Recently, we discovered that murine embryonic stem cells express seven of eight mammalian Src family kinases (SFKs), and that their activity and gene expression patterns change in distinct ways during differentiation to embryoid bodies. Pharmacological inhibition of overall Src kinase activity reversibly blocks differentiation of ES cells while maintaining pluripotency. These findings suggest critical roles for individual Src family members in the early fate determination of ES cells. This project will address the broad hypothesis that individual SFKs play necessary, non-redundant roles in ES cell proliferation, self-renewal and germ layer formation. In Aim 1 SFK variants with functionally silent mutations that render them uniquely sensitive to inhibition by small molecules will be used to replace both alleles of the corresponding endogenous kinase genes in ES cells. The resulting knock-in cell lines will then be analyzed in terms of proliferation, self-renewal and embryoid body formation in the presence and absence of the selective inhibitor. Complementary experiments in Aim 2 will utilize SFK mutants in which sensitivity to broad- spectrum SFK inhibitors is engineered out. These mutants will be tested for their ability to rescue the growth and differentiation block induced by SFK inhibitors. In Aim 3, comparative proteomic analyses will be performed to identify unique proteins that bind to recombinant SFK SH3-SH2 domains in ES cells and embryoid bodies. Differences in partner protein binding to this regulatory region of SFKs may account for biological differences observed in terms of SFK control of self-renewal and differentiation. Successful completion of this work will shed new light on cytoplasmic tyrosine kinase signaling pathways that control ES cell fate, and has the potential to identify individual SFKs and other intracellular signaling molecules as pharmacological targets for manipulation of stem cell behavior with small molecules. Public health relevance: This work is focused on molecular mechanisms that control the growth and differentiation of embryonic stem cells. Understanding the cellular machinery that regulates these critical cellular events is the first step towards identification of pharmacological agents that will permit expansion and differentiation of ES cells in vitro. The ultimate utility of stem cells in clinical medicine depends upon safe and effective methods for manipulating their renewal and differentiation in culture.